FRET-Based Molecular Dynamics Simulation of Membrane-Receptor Activation
EMSL Project ID
25400
Abstract
This proposal is a continuation of research pursued under EMSL User Proposals #6494 and #6493. Membrane receptors are activated upon the binding of small ligand molecules to specific sites in their extracellular domain, and in turn, they initiate molecular cascades in the intracellular environment, ultimately dictating the cellular response to the extracellular cue. However, the exact mechanisms by which membrane receptors execute their function are not completely understood. Here we propose to acquire live cell fluorescence resonance energy transfer (FRET) measurements that will guide molecular dynamics simulations to gain a new understanding of the interactions and conformational motions that underlie the function of specific receptor tyrosine kinases. We will focus on the epidermal growth factor (EGF) receptor and its dimerization partner, human epidermal growth factor-2 (HER2), expressed in human mammary epithelial (HME) cells. The EGF receptor and HER2 play important roles in the normal function of many cells and tissues, yet the nature of their interactions and mechanisms of their activation are still unclear. These studies will directly contribute to EMSL Biological Interactions and Dynamics science theme by deciphering the interactions and conformational dynamics that underlie the activation of membrane receptors in response to external cues using integrations of experimental and computational approaches. Most of our understanding of protein conformational motions has been only inferred from static structures or images, biochemical studies of proteins isolated from their cellular environment, or from physiological measurements of protein function endpoints. Molecular dynamics simulations have contributed to our understanding of the structure-function relationships by adding the time dimension. The simulation techniques, however, have been challenged by the large size of membrane proteins and their complexes, and also by the limited time scales of the techniques, which are often shorter by several orders of magnitude than the time scale needed to observe the whole process with sufficient structural details. Here we propose to take advantage of EMSL capabilities in high resolution live cell FRET imaging and spectroscopy to provide direct, rather than inferred, distance measurements using FRET within and between receptors undergoing conformational motions in the membrane of a living cell. The measurements in the starting and ending conformational states will direct the application of minimal forces during molecular dynamics simulations to guide the change between the two states. Such an approach will enable the simulation of the whole process in a contracted time scale. A growing body of evidence suggests that the EGF receptor can exist in the membrane as a preformed dimer that spontaneously occurs even in the absence of the ligand. However, the conformational motions by which a ligand could activate a preformed dimer are unclear. We will build on our previous work using EMSL methods for precise quantification of FRET efficiencies, which will to guide molecular dynamics simulations, to gain a dynamic view of the activation process. We will increase the accuracy in mapping the FRET measurements to the simulated structures using high affinity small fibronectin-scaffold domains instead of Fab fragments and include domain IV of the receptor in our measurements. Using this approach we will also elucidate the conformational motions underlying the activation of the EGF receptor-HER2 heterodimer. Considering the critical role of HER2 in the etiology of disease, a detailed understanding of the mechanism underlying its activation is an important step toward developing new and better therapeutic approaches. The studies above will gain new understanding of the cellular responses to external cues via the activation of membrane receptors.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-06-15
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members